The bone vitamin K dependent protein, osteocalcin, is an osteoblast specific product with specialized calcium binding properties to hydroxyapatite. Its measurement in the circulation is widely used as a non-invasive parameter of bone turnover, yet its precise function remains elusive. We have evidence to postulate a role for osteocalcin in mediating osteoclast differentiation and activation in several in vivo and in vitro model systems developed for the study of bone resorption. We have also shown osteocalcin can directly promote fusion of progenitor cells into multinucleated tartrate resistant acid phosphatase positive (TRAP) cells that develop a ruffle border membranes, characteristic features of osteoclasts. Our long range goal is to define the precise mechanism by which osteocalcin participates in the regulated resorption of bone mineral. In the current proposal our efforts are initially to define the specificity of osteocalcin in the early stages of recruitment of progenitor cells and their fusion to multinucleated activated osteoclasts using the in vivo implant system and in vitro culture studies with bone marrow progenitor cells. Secondly we will define specific regions of the osteocalcin molecule that are requisite for promoting osteoclast differentiation site directed mutagenesis of the rat osteocalcin gene. Those amino acid which participate in presumed function and precise structural folding of the molecule when associated with hydroxyapatite crystals will be altered and the molecules tested in the in vivo implant model and in vitro bone marrow cultures. Thirdly the molecular defect associated with osteocalcin-deplete bone in osteoclast differentiation will be identified by, +/- and subtractive hybridization screening of cDNA libraries generated from functional osteoclast generated to normal bone particles and to the multinucleated cell surrounding osteocalcin-deplete bone implants. We will characterize the genes expressed during osteoclast differentiation that are mediated by osteocalcin. These proposed studies will define a molecular role for osteocalcin in the differentiation of progenitor cells to a fully functioning osteoclast and in doing so we will better understand control mechanisms of normal bone resorption by bone matrix components. Knowledge of the structure function relationships of the osteocalcin molecule to induction of osteoclast differentiation and bone resorption could provide a rationale for therapeutic intervention of bone resorption osteoporosis and in diseases associated with high bone turnover.